Characterization of MVP and VPARP assembly into vault ribonucleoprotein complexes.
Characterization of MVP and VPARP assembly into vault ribonucleoprotein complexes.
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DOI：
10.1016/j.bbrc.2004.11.006
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发表时间：
2004-12
影响因子：
3.1
通讯作者：
Chun-lei Zheng;T. Sumizawa;Xiao-Fang Che;S. Tsuyama;T. Furukawa;M. Haraguchi;Hui Gao;T. Gotanda;Hei-Cheul Jueng;F. Murata;S. Akiyama
中科院分区：
文献类型：
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作者：
Chun-lei Zheng;T. Sumizawa;Xiao-Fang Che;S. Tsuyama;T. Furukawa;M. Haraguchi;Hui Gao;T. Gotanda;Hei-Cheul Jueng;F. Murata;S. Akiyama
穹窿是由三种蛋白质组成的桶形细胞质核糖核蛋白颗粒：主要穹窿蛋白（MVP），穹窿聚（ADP-核糖）聚合酶（VPARP）和端粒酶相关蛋白1，以及一个或多个小的非翻译RNA。迄今为止，人们对拱顶装配过程或拱顶构件的稳定性知之甚少。在这项研究中，我们分析了MVP和VPARP的生物合成，以及它们在人ACHN肾癌细胞穹窿颗粒内的半衰期。使用免疫沉淀测定，我们发现新合成的MVP需要超过4小时才能掺入拱顶颗粒中，但生物合成的VPARP在1.5小时内完全掺入拱顶中。一旦加入拱顶复合体，MVP和VPARP都非常稳定。在大肠杆菌中单独表达人MVP导致形成具有独特拱顶形态的颗粒。缺乏聚（ADP-核糖）聚合酶活性的VPARP的C-末端区域与MVP颗粒共沉淀。这表明VPARP的活性对于与MVP-自组装拱顶样颗粒的相互作用不是必需的。总之，我们的研究结果提供了一个深入了解生理穹窿组装的潜在机制。
Vaults are barrel-shaped cytoplasmic ribonucleoprotein particles composed of three proteins: the major vault protein (MVP), the vault poly(ADP-ribose)polymerase (VPARP), and the telomerase-associated protein 1, together with one or more small untranslated RNAs. To date, little is known about the process of vault assembly or about the stability of vault components. In this study, we analyzed the biosynthesis of MVP and VPARP, and their half-lives within the vault particle in human ACHN renal carcinoma cells. Using an immunoprecipitation assay, we found that it took more than 4h for newly synthesized MVPs to be incorporated into vault particles but that biosynthesized VPARPs were completely incorporated into vaults within 1.5h. Once incorporated into the vault complex, both MVP and VPARP were very stable. Expression of human MVP alone in Escherichia coli resulted in the formation of particles that had a distinct vault morphology. The C-terminal region of VPARP that lacks poly(ADP-ribose)polymerase activity co-sedimented with MVP particles. This suggests that the activity of VPARP is not essential for interaction with MVP-self-assembled vault-like particles. In conclusion, our findings provide an insight into potential mechanisms of physiological vault assembly.